Identification and partial characterization of proteolytic activity of Enterococcus faecalis relevant to their application in dairy industry

Abstract

Bacteria of the genus Enterococcus are lactic acid bacteria (LAB), which occur ubiquitous in many traditional fermented foods, especially artisanal cheeses, playing positive role in the development of cheese flavor. Moreover, several enterococci are successfully used as a pharmaceutical probiotic and some of them are able to produce bacteriocin and bioactive peptides, thanks to which the possibilities of application of enterococci in dairy technology and biotechnology are increased.
 The aims of the study were to investigate the proteolytic potential and identify the key enzymes of proteolytic system of Enterococcus faecalis isolated from artisan Polish cheeses. An extracellular - secreted (E) and a cell envelope proteinase (CEP) were isolated and enzyme activity depending on bacterial growth phase was evaluated. CEP showed a higher protease activity than E and this fraction has been purified 70-fold by a method including precipitation, diafiltration and gel filtration chromatography. The molecular mass of the enzyme has been estimated to be ~25 kDa by SDS-PAGE. Maximum enzyme activity of the proteinase has been observed at pH 6,9 and 37 ºC. The enzyme was able to hydrolyze: casein, bovine serum albumin, α-lactalbumin, β-lactoglobulin, but not Leu-pNa. The results of zymography, SDS- PAGE and LC-MS-MS/MS data allowed us to identify the key enzymes of proteolytic system of E. faecalis as coccolysin and glutamylendopeptidase.
 To asses microbiological safety of the tested strain, the evaluation of the presence of virulence factors and antibiotic susceptibility was also conducted.

Metabolism Characteristics of Lactic Acid Bacteria and the Expanding Applications in Food Industry

“…In Lentilactobacillus hilgardii, the decarboxylation reaction of histidine and tyrosine also contributes to the acid resistance of the bacteria (Lamberti et al, 2011). Levilactobacillus brevis, Latilactobacillus curvatus, (Biscola et al, 2018;Kordesedehi et al, 2018;Worsztynowicz et al, 2019) Different bioactive peptides and the bioactivity diversity can be increased by editing the proteolytic system of Lactococcus lactis…”

“…Especially in the fermentation process of dairy products, lactic acid bacteria can degrade casein, thereby reducing the allergenicity of dairy products ( Iwamoto et al, 2019 ). For example, certain strains of Enterococcus faecium isolated from fermented milk and cheese can express metalloproteases or cell envelope proteinase (CEP) ( Genay et al, 2009 ), etc., which can effectively hydrolyze casein in milk ( Biscola et al, 2018 ; Kordesedehi et al, 2018 ; Worsztynowicz et al, 2019 ). Lactic acid bacteria have made significant contributions not only in dairy products, but also in other fermented foods, such as fermented fruit and vegetable products and fermented grain products.…”

The Many Faces of Enterococcus spp.—Commensal, Probiotic and Opportunistic Pathogen

“…Enterococcus strains also display proteolytic activity (producing of extracellular proteinases, intracellular peptidases, and transport enzymes) [ 158 , 159 ] and play an important role in bacterial growth [ 158 ]. Some of them, including extracellular-secreted (E) or cell envelope proteases are used in the fermentation of dairy products) [ 142 , 160 ].…”

Characteristics of the Proteolytic Enzymes Produced by Lactic Acid Bacteria

“…Easily absorbable dairy products are produced for sick people and children. The exogenous proteolytic enzymes used for cheese production are very important [6,[98][99][100][101]. Some microbial proteases produced by LAB may replace chymosin in cheese production [102].…”

“…Some microbial proteases produced by LAB may replace chymosin in cheese production [102]. E. feacalis is able to produce active protease, which indicates the possibility of its application to support the breakdown and release of bioactive peptides from whey protein [99]. LAB (E. faecalis VB43) also produce enzymes that are capable of hydrolysing allergenic proteins in milk, so they have good potential for the manufacture of hypoallergenic dairy products [103].…”